Adiponectin (see Non-Patent Documents 1 to 4) is a secretion-type protein expressed specifically and abundantly in white adipose tissue. Adiponectin is a plasma protein (about 30 kDa) belonging to the C1q family and is composed of 244 amino acids.
Adiponectin possesses a trimeric structure of triple helix, in which each monomer is formed of an N-terminal collagen-like domain with multiple Gly-X-Y repeats and a C-terminal globular domain. Also, it has been reported that, in blood, a plurality of trimers are linked to one another to form higher order products (hereinafter may be referred to as “various multimers”).
In recent years, adiponectin has been reported to exist in human blood at a high level of 5 to 10 μg/mL, and exerts a variety of physiological activities. In particular, adiponectin suppresses growth of smooth muscle cells and prevents monocytes from adhering onto endothelial cells. From these findings, adiponectin is considered to have anti-arteriosclerosis effect (Non-Patent Document 5). Moreover, from the findings that when adiponectin is administered to a mouse suffering from type 2 diabetes or lipoatrophic diabetes, insulin resistance is reversed and hyper-FFA (free fatty acid) in blood and hyper-TG (triglyceride) in blood are alleviated, adiponectin is reported to function as an insulin-sensitive hormone and to exhibit ameliorating effect on diabetes (Non-Patent Document 6). It is also reported that renal failure patients who show low blood adiponectin levels have a high risk of complications of cardiovascular diseases and show low survival rates, and that, in a study performed on native Americans of the Pima tribe who are known to develop insulin resistance and type 2 diabetes at a high incidence, onset of type 2 diabetes is suppressed among subjects showing high blood adiponectin (Non-Patent Document 7).
The above findings suggest the possibility that adiponectin might be an endocrine factor responsible for linking the excessive accumulation of visceral fat directly with onset of insulin resistance. Therefore, blood adiponectin level is considered a predictive factor for the onset of diabetes or arteriosclerosis, and measurements of such levels are expected to serve as a useful indicator of lifestyle-related diseases.
According to a method for determining the total amount of various multimers of adiponectin contained in a blood sample, a sample is boiled in the presence of sodium dodecylsulfate (SDS) to expose antibody-recognizing sites of various multimers which have been hidden stereostructually, and then immunoassay is performed (Patent Document 1). However, this method has some problems in that it requires an apparatus for boiling treatment (100° C.) and it is also actually difficult to make itself available to automation of two steps; i.e., boiling treatment and subsequent immunoassay.
A kit called “HUMAN ADIPONECTIN RIA KIT” (Cat. #HADP-61HK) is commercially available (LINCO RESEARCH, INC.). However, given that the kit utilizes the two-antibodies/PEG method, in which 125I-labelled mouse adiponectin and human adiponectin are competed and anti-adiponectin polyclonal antibody is used for capture, it should be noted that handling of this kit is cumbersome, and in addition, there remain concerns about safety, specificity, and quality of the reagents. In order to make it possible that the specificity in the above method established on the basis of the competitive reaction continues to be constant, there is need for the conditions under which the reactivity of anti-adiponectin polyclonal antibody against the 125I-labelled mouse adiponectin and various human adiponectin multimers continue to be constant. However, as described before, a biological sample contains various multimers in a mixed state, and the proportions of the respective multimers vary. Thus, this kit essentially involves the problem that total adiponectin cannot be measured accurately.
In addition, there is a prior art document disclosing a monoclonal antibody which recognizes non-denatured adiponectin having a specific stereostructure that has not been modified by any denaturation treatment with, for example, SDS or heat (see Patent Document 2, the adiponectin of this type is referred to as native adiponectin in this reference), and an assay method utilizing the monoclonal antibody (Patent Document 2). However, this method has the problem that total adiponectin of a biological sample cannot be measured accurately as it contains various multimers at varying proportions because the form of adiponectin present in a sample (for example, the number of trimers and trimer aggregation condition) affects reactivity of adiponectin with the above-mentioned monoclonal antibody.
The structural form of adiponectin has been investigated with respect to a recombinant, though not in a biological sample. According to such investigation, when adiponectin is treated with dithiothreitol (DTT) at low pH (Non-Patent Document 8) or with trypsin (Non-Patent Document 9), the structural form thereof changes. However, there is no information about results of an immunoassay of the adiponectin subjected to such a treatment.
As described above, in order to immunologically determine the total adiponectin level of a sample, the sample must be subjected to a pretreatment process to thereby attain a uniform reactivity between each of the multimer species (a trimer and various multimers composed of trimers) and the antibody employed. However, there has been no convenient method whose two steps; i.e., the pretreatment step and the immunoassay step, can be automated.    [Patent Document 1] Japanese Patent Application Laid-Open (kokai) No. 2000-304748    [Patent Document 2] PCT International Publication WO03/016906    [Non-Patent Document 1] Scherer P. E., et al., J. Biol. Chem. 270, 26746-26749, 1995    [Non-Patent Document 2] Hu E., et al., J. Biol. Chem. 271, 10697-10703, 1996    [Non-Patent Document 3] Maeda K., et al., Biochem. Biophys. Res. Commun. 221, 286-289, 1996    [Non-Patent Document 4] Nakano Y., et al., J. Biochem. 120, 803-812, 1996    [Non-Patent Document 5] Ouchi N, et al., Circulation, 102, 1296-1301, 2000    [Non-Patent Document 6] Yamauchi T, et al., Nature Med. 7, 941-946, 2001    [Non-Patent Document 7] Lindsay R. S., et al., Lancet, 360, 57-58, 2002    [Non-Patent Document 8] Utpal B. Pajvani, et al., J. Biol. Chem. 278, 9073-9085, 2003    [Non-Patent Document 9] Fruebis, J., et al., Proc. Natil. Acad. Sci. 98, 2005-2010, 2001